臺灣博碩士論文加值系統

Current understanding of T cell receptor (TCR) triggering is based on the segregation of membrane tyrosine phosphatases having large ectodomains upon engagement between T cells and antigen presenting cells, thereby creating a kinases/phosphatases imbalance to phosphorylate immuno-receptor tyrosine-based activation motifs (ITAMs) in CD3 proteins associated with engaged TCRs. We recently observed that elongated OKT3 anti-CD3 single-chain antibodies (scFv) expressed on 3T3 fibroblasts can effectively trigger TCR signaling, inconsistent with a requirement for CD45 segregation from engaged TCRs. Here, we show that TCR triggering by elongated ligands depends on affinity rather than dimension, and CD45 segregation is not a mandatory step to initiate TCR triggering. We created elongated scFv with defined dimensions expressed on the surface of 3T3 APCs and or distributed over planar lipid bilayers to track CD45 movement by TIRF microscopy. We found that elongated scFv (OKT3-CD43) was unable to segregate CD45 from the ligated TCR microclusters, but inconsistent with the KS model, was able to induce T cell calcium flux, and Zap70 phosphorylation. However, within minutes of triggering, CD45 started to segregate partially from the TCR microclusters or pZap70, indicating that CD45 segregation follows rather than initiates TCR triggering. Using a mutated OKT3 scFv that showed low affinity to CD3ε (OKT3MA), we proved that TCR triggering is more dependent on ligand affinity rather than the ligand dimensions and CD45 physical segregation; short, low affinity scFv (OKT3MA-BGP) was able to trigger TCR similarly to wild type scFv (OKT3-BGP), while elongated low affinity OKT3MA scFv (OKT3MA-CD43) was unable to trigger TCR, nor segregate CD45. Longer follow up of T cells showed proliferation, IL-2 and IFN-γ secretion by the short ligands (high and low affinities) while only high affinity elongated ligand was able to produce productive T cell activation.

Abstract ……………………………………………………………………………….. i
Table of Contents ……………………………………………………………………... ii
List of Figures ………………………………………………………………………… v
List of Abbreviations ………………………………………………………………….. vii
I. Introduction ………………………………………………………………………... 1
II. Background ……………………………………………………………………….. 3
1. T Lymphocytes ………………………………………………………………….. 3
2. Elements for TCR Triggering …………………………………………………… 4
2.1. Ligands …………………………………………………………………….. 5
2.2. T Cell Receptor …………………………………………………………….. 5
2.3. Co-Receptors ……………………………………………………………….. 6
2.4. Adhesion Molecules ………………………………………………………... 7
2.5. Kinases and Phosphatases …………………………………………………… 8
2.6. Other Signaling Proteins ……………………………………………………. 11
3. TCR Triggering …………………………………………………………………… 12
4. Immunological Synapse …………………………………………………………… 13
5. Ligand Properties ………………………………………………………………….. 14
5.1. Binding Site …………………………………………………………………. 14
5.2. Affinity ………………………………………………………………………. 15
5.3. Dimension …………………………………………………………………… 16
6. TCR Triggering Models …………………………………………………………… 16
6.1. Serial Triggering Model …………………………………………………… 17
6.2. Kinetic Proof-Reading Model ……………………………………………… 18
6.3. Occupancy Model (Affinity Model) ……………………………………….. 18
6.4. Co-receptor Heterodimerization Model ……………………………………. 19
6.5. Pseudodimer Model ………………………………………………………... 19
6.6. Conformational Change Model ……………………………………………. 20
6.7. Kinetic Segregation Model ………………………………………………… 21
6.8. Lipid Raft Model…………………………………………………………… 22
6.9. Receptor Deformation Model ……………………………………………… 23
7. Mechanical Forces Driving TCR Triggering …………………………………….. 24
III. Study Objective …………………………………………………………………… 28
IV. Material and Methods …………………………………………………………….. 29
V. Results ………………………………………………………………………………. 39
1. Ligands Elongation on 3T3 APCs ……………………………………………….. 39
1.1. Generation of APCs Expressing Ligands with Different Affinities and Dimensions ………………………………………………………………... 39
1.2. Ligand Affinity Determines Potency of TCR Triggering …………………. 40
1.3. Surface Density is not the Major Factor Driving the Dimension-Independent T Cell Activation by Membrane-Tethered OKT3 Anti-CD3 ScFv ………. 40
2. Ligand Elongation on Lipid Bilayers …………………………………………….. 41
2.1. Production of Soluble Anti-CD3ε ScFv Ligands …………………………... 41
2.2. Lipid Bilayer-Coated Glass Beads Decorated with ScFv Ligands Show a Similar Trend of T Cell Activation as 3T3 APCs …………………………. 42
2.3. Ligand Elongation on Lipid Bilayers Has No Effect on Jurkat T Cell Binding …………………………………………………………………….. 43
2.4. Jurkat T Cells Respond by Releasing Calcium upon TCR Triggering …….. 44
2.5. TCR Triggering is Initiated by Elongated High Affinity Ligand Without Clear CD45 Segregation …………………………………………………… 45
2.6. CD45 is Efficiently Segregated from pZap70 by Short Ligands and Partially from Elongated High Affinity Ligands …………………………………… 46
2.7. Elongated Non-Activating Ligand Accumulates CD45 Segregation with Time Without T Cell Activation …………………………………………… 47
VI. Discussion ………………………………………………………………………… 68
VII. References ……………………………………………………………………….. 80
VIII. Appendix ………………………………………………………………………… 93
(Chen BM, Al-Aghbar MA)*, Lee CH, Chang TC, Su YC, Li YC, Chang SE, Chen CC, Chung TH, Liao YC, Lee CH, Roffler SR, 2017. The affinity of elongated membrane tethered ligands determines potency of TCR triggering. Front. Immunol.8:793
* equal contribution …………………………………………………………………….93

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